The comprehensive quantitation of biomolecules and fragments of the biomolecules in a sample, such as proteins expressed by a genome, cell, tissue or organism, is a complex task.
In recent years, mass spectrometry has made great technological progress and is increasingly broadly applied (R. Aebersold and M. Mann, Nature 422 (6928), 198 (2003), B. F. Cravatt, G. M. Simon, and J. R. Yates, 3rd, Nature 450 (7172), 991 (2007)). However, accurate quantitation of the entire complement of biomolecules such as proteins expressed by a genome, cell, tissue or organism, e.g., a tumor proteome, by high resolution mass spectrometric methods is still in its infancy (F. Bertucci, D. Birnbaum, and A. Goncalves, Mol Cell Proteomics 5 (10), 1772 (2006), J. M. Koomen, E. B. Haura, G. Bepler et al., Mol Cell Proteomics 7 (10), 1780 (2008)).
A primary difficulty has been to quantify a representative number of biomolecules, which is a prerequisite for obtaining reproducible results, and for studying disease related biomolecules, which are often low abundant, such as cancer relevant proteins.
Molecular classification of certain disease states, e.g., tumors, can aid in patient segregation, selection of optimal treatment modalities and prediction of outcome. Measuring transcriptome levels with microarrays has shown promise for this application and is starting to be clinically applied (T. R. Golub, D. K. Slonim, P. Tamayo et al., Science (New York, N.Y 286 (5439), 531 (1999), X. Li, R. J. Quigg, J. Zhou et al., Current genomics 9 (7), 466 (2008)).
Stable isotope labeling by amino acids in cell culture (SILAC) is very accurate and robust, which makes it a valuable tool for quantifying proteomes (S. E. Ong, B. Blagoev, I. Kratchmarova et al., Mol Cell Proteomics 1 (5), 376 (2002), S. E. Ong and M. Mann, Nature protocols 1 (6), 2650 (2006)). For example, Chowdhury et al. (Rapid Communications in Mass Spectrometry 9: 563-569 (1995) used mass spectrometry and isotopically labeled analogs to investigate the molecular weight of truncated mature collagenase, and Stocklin et al. (Diabetes 46: 44-50 (1997) investigated human insulin concentration in serum samples that had been extracted and purified. Neither one discusses the determination of the quantity of biomolecules without prior isolation of the biomolecules, let alone determining the absolute quantity of a plurality of biomolecules comprised in a sample, e.g., the entire complement of biomolecules in such a sample.
Because SILAC requires complete metabolic labeling of the entire proteome, it has usually been limited to cell culture and is thought to be unsuitable for clinical samples. In recent years, a few studies have broadened the scope of SILAC. The Neuro2A cell line has been metabolically labeled and compared to total mouse brain (Y. Ishihama, T. Sato, T. Tabata et al., Nature Biotechnology 23 (5), 617 (2005)). A total of 602 proteins were quantified, albeit with up to 10-fold ratios between cell line and tissue. Such high ratios between sample and internal standard make accurate quantification difficult because the lower abundant peptide in the SILAC-pair may be close to the noise level.
Proteins from cultured primary hepatocytes 2 isolated from mouse liver using SILAC-labeled Hepa1-6 cells have been recently quantified (S. Pan, L. Cheng, J. T. White et al., Omics 13 (4), 345 (2009)). Furthermore, a mouse has been previously SILAC-labeled, but this method is not applicable to human subjects (M. Kruger, M. Moser, S. Ussar et al., Cell 134 (2), 353 (2008)).
There is therefore a need for a straightforward and rapid method for the quantitation of one or a plurality, e.g., the entire complement of biomolecules (e.g., proteins, oligonucleotides, etc.) in a sample, such as a human sample and particularly a human tissue sample, which furthermore allows to rapidly quantify a plurality of biomolecules, e.g., a part of, or the entire complement of biomolecules in such a sample, such as proteins expressed by a genome, cell, tissue or an organism.
The above object is solved by the methods according to the present invention as described and claimed herein.